Biomaterial, injectable implant comprising it, its method of preparation and its uses

ABSTRACT

This invention relates to a biomaterial comprising an injectable bioresorbable polysaccharide composition wherein the polysaccharide may be succinochitosan glutamate. This invention also relates to a biomaterial comprising an injectable bioresorbable polysaccharide composition in which resorbable particles may be in suspension, the said particles comprising or consisting essentially of chitin and/or chitosan, which may be free of any additional formulation modifying agents, and a process for manufacturing the same. This invention also relates to a medical device including said biomaterial and to a method of repair or treatment including the filling of cavities in the human face or body.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/677,389, filed Feb. 21, 2007, which claims priority to French PatentApplication No. FR 06 01657, filed Feb. 24, 2006, the contents each ofwhich is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates to the field of biomaterials forimplantation in the human or animal body. More particularly, the presentinvention relates to an implantable biomaterial, which may comprisechitin and/or chitosan. The biomaterial of this invention may be in theform of a gel, and may be injected, in particular by the subcutaneous orintradermal route, to form an implant. This implant has the benefit ofbeing bioresorbable.

BACKGROUND OF INVENTION

Experts in the field are familiar with various injectable implants. Forexample, silicon gels (or silicon oils) are well-known, but these gelshave the inconvenience of not being biodegradable. Moreover, silicon isoften the cause of chronic inflammation, granuloma formation and evendelayed allergic reactions. Collagen suspensions have also been verywidely used over the past ten years. However, collagen generally is ofbovine origin, which is undesirable for health and generally subject toadditional regulatory requirements. Attempts to re-implant fatty cellsremoved from the patients themselves are also reported. However, theduration of the filling effect is generally less than the patient wouldlike.

Other implants have been used, comprising a gelatine or collagensolution including, in suspension, polymethyl methacrylate (PMMA)microspheres having a diameter of 20 to 40 μm. PMMA, however, is notbiodegradable and the gelatine or collagen solution is generally derivedfrom bovine sources.

EP 0 969 883 describes an implantable gel including L-PLA (polylacticacid) microspheres with a diameter of 20 to 40 μm suspended in a carboxymethylcellulose gel (CMC). This gel is injectable and can be supplied ina sterile syringe. This product shows an acceptable efficacy but maypresent poor syringability (clogging of the required low-diameterneedles may be noted) and a biodegradability which is too slow for someof the desired applications. The particles have the tendency toaggregate in the packaging, in particular in a syringe, makinginjections difficult and leading to inconsistent results.Non-homogeneous distribution of the particles in the injection area mayactually be observed in patients. The expected aesthetic result istherefore not achieved and areas overloaded with particles are noted,sometimes adjacent to areas free of particles. The very long resorptiontime of the PLA (having a high molecular weight) may be of severalyears, which may also lead to inflammatory reactions in the long run.

There is therefore a real need for new biomaterials which do not havethe drawbacks of the prior art, and particularly biomaterials which areuseful as immediate filler materials, able to generate fibrosis and alsocapable of being resorbed to avoid chronic inflammatory reactions orrejection in the long run.

SUMMARY OF INVENTION

The inventors have found a biomaterial satisfying this need, whichcomprises an injectable composition, preferably in the form of a gel ofchitin or chitosan, such as for example a succinochitosan glutamate gel,preferably including particles in suspension in the composition, saidparticles comprising chitin and/or chitosan. The biomaterial of theinvention is bioresorbable, and when particles are in suspension theyare bioresorbable, as well. The resorption time of the gel may bedifferent from the resorption time of the particles.

According to the invention, the biomaterial of the invention produces afilling effect, resulting from the injected volume of composition. Animportant goal of the biomaterial of the invention is to induce fibrosisand tissue, especially neo-tissue (dermis), formation.

DESCRIPTION OF FIGURES

FIG. 1 is a photo showing the distribution of chitin particles for asuccinochitosan glutamate gel containing 1% chitin particles. The photoof the biomaterial according to the invention, taken using an OLYMPUS®optical microscope, confirms that the particles are distributedhomogeneously throughout the gel, naturally remaining in suspension dueto the surfactant properties of chitosan, without the need of additionalsurfactants.

DETAILED DESCRIPTION OF THE INVENTION

Fibrosis is induced by biomaterial, which means by the composition andby the particles present in the biomaterial of the invention. When thebiomaterial is injected, it is perceived as foreign bodies and the bodyresponds to this attack by connective tissue hyperplasia, withproliferation of fibroblasts developing from collagen(neo-collagenesis). Fibrosis reaction induced by injecting thebiomaterial of the invention may occur between 15 days and 3 weeks afterinjection.

Inducing fibrosis by injecting the biomaterial of the invention, isaimed to create natural filling tissue which will replace thebiomaterial when it is resorbed. It is therefore desirable that theparticles, which may be considered as being principally responsible forinducing the fibrosis, be resorbed once they no longer fulfill theirfunction of inducing fibrosis, preferably within a period of 1 to 6months.

Thus, the biomaterial according to the invention, partly because of thenature of its composition and partly because of the presence ofparticles, proposes a technical solution for patients in need forimplantable filling material, and the product biodegradation andresorption time of the biomaterial may be adapted to the specific needsof the patients, for example by adjusting the amount of particles in thecomposition, thus avoiding the drawbacks of the prior art products.

Injecting a high amount of the biomaterial of the invention in a singleinjection may not be the optimal method of treating patients in need ofsaid biomaterial, since the enhancement of the tissue (e.g., dermis) maynot depend on the amount of biomaterial injected in a single injection;it may be preferred to carry out several injections, which may bedistant of a few weeks, for example two months. This embodiment aims atletting the biomaterial almost totally resorb before injecting newbiomaterial.

In the present invention, by syringability is meant the ease ofinjection of the biomaterial; syringability generally may be a functionof viscosity and other rheological properties of the biomaterial and ofthe size of the particles included within the biomaterial and ofdiameter of the needle of the syringe. By chitin, is meant a linearpolysaccharide of beta-1.4-N-acetyl-D-glucosamine. By chitosan is meanta linear polysaccharide composed of randomly distributed linkedbeta-1.4-linked N-acetyl-D-glucosamine (acetylated unit) andD-glucosamine (deacetylated unit). The degree of deacetylation ofchitosan may be determined by NMR spectroscopy.

By chitosan derivative, is meant any chitosan salt or acid-derivedchitosan, chitosan glycolate, chitosan lactate, chitosan succinate,hydroxyalkyl chitosan, chitosan acetate, chitosan glutamate and morepreferably succinochitosan glutamate.

According to a preferred embodiment of the invention, the biomaterial ofthe invention comprises or consists of an injectable bioresorbablepolysaccharide composition, preferably in the form of a gel, includingresorbable particles in suspension within the composition, saidparticles comprising chitin and/or chitosan.

In one embodiment, the polysaccharide is chitosan or a derivativethereof, preferably having a degree of deacetylation of about 30 toabout 95%, preferably about 70 to about 90%, more preferably about 75 toabout 85%, even more preferably about 80 to about 85%, and mostpreferably about 85%.

Advantageously, the molecular weight of the chitosan or the chitosanderivative of the gel composition or used to make the chitin or chitosanparticles is of about 10 000 to about 500 000 D, preferably about 30 000to about 100 000 D, more preferably about 50 000 to about 80 000 D.

According to an embodiment, the biomaterial includes in its compositionabout 0.1 to about 20%, preferably about 1 to about 20% w/w, morepreferably about 1 to about 12% w/w, even more preferably about 1 toabout 10%, most preferably about 1 to about 5% w/w of polysaccharidewhich is a chitosan or a chitosan derivative. In a specific embodiment,when the polysaccharide is a chitosan derivative, the composition of thebiomaterial may include about 0.1 to about 20% of polysaccharide.According to a particularly preferred embodiment the chitosan derivativeis succinochitosan glutamate.

According to an embodiment, the biomaterial of the invention comprisesan injectable bioresorbable polysaccharide composition wherein thepolysaccharide is succinochitosan glutamate. Advantageously, thesuccinochitosan glutamate has a degree of deacetylation of about 30 toabout 95%, preferably about 70 to about 90%, more preferably about 75 toabout 85%, even more preferably about 80 to about 85%, and mostpreferably about 85%. According to an embodiment, the succinochitosanglutamate has a molecular weight of about 10 000 to about 500 000 D,preferably about 30 000 to about 100 000 D, more preferably about 50 000to about 80 000 D. According to an embodiment, the composition comprisesabout 0.1 to 20%, preferably 1 to 10%, more preferably 1 to 5% w/wsuccinochitosan glutamate by weight of the total composition.Advantageously, the biomaterial of the invention is a gel. Thesuccinochitosan glutamate may be derived from chitosan of animal orvegetal origin. Advantageously, the succinochitosan glutamate used tomanufacture the biomaterial of the invention is derived from GMP-gradechitosan.

In another preferred aspect of the invention, the biomaterial is achitosan or chitosan derivative gel including chitin particles.

According to a most preferred embodiment of the invention, thebiomaterial is a gel of succinochitosan glutamate, including chitinparticles in suspension within the gel.

According to an embodiment, the chitosan used for manufacturing thebiomaterial of the invention may be either of animal or vegetal origin.The use of a chitosan of animal origin, and more particularlycrustaceans (prawn shells) or squids is of economic benefit. The use ofa product of vegetal origin, and more particularly fungal, is generallybetter appreciated by consumers. Thus, according to a preferredembodiment, the chitosan used in the biomaterial of the invention, isextracted from fungi, such as for example Mucoralean strains, Mucorracemosus and Cunninghamella elegans, Gongronella butleri, Aspergillusniger, Rhizopus oryzae, Lentinus edodes, Pleurotus sajo-caju, morepreferably Agaricus bisporus. According to another embodiment, thechitosan was produced from two yeasts, such as, for exampleZygosaccharomyces rouxii and Candida albicans.

According to a particular embodiment of the invention, the particlesincluded within the biomaterial of the invention contain or consistessentially of chitin and/or chitosan which are either of animal orvegetal origin. The particles may also be made of, or include, a mixtureof chitin and chitosan. According to an embodiment, these particles mayconsist solely of chitin or solely of chitosan. According to anembodiment, the chitosan used to make the particles may have a degree ofdeacetylation of about 30 to about 95%, preferably about 70 to about90%, more preferably about 75 to about 85%, even more preferably about80 to about 85%, and most preferably about 85%. Advantageously, thechitosan used to make the particles of the invention may be of GMPgrade. According to a preferred embodiment, the particles are of chitinobtained by reacetylation of a GMP grade chitosan. According to apreferred embodiment, the biomaterial of the invention is essentiallyfree of endotoxins. According to another embodiment, the biomaterialincludes deproteinized chitin particles essentially free of endotoxins.

According to an embodiment, the particles included within thebiomaterial of the invention have a bioresorption time of to 6 months.According to an embodiment, the chitin-only particles with abioresorption time of 1 to 3 months, and chitosan-only particles have abioresorption time of 1 to 4 months.

According to another embodiment of the invention, the amount ofparticles in the biomaterial of the invention may be of about 0.1 toabout 10% w/w, preferably of 1 to 5% w/w, more preferably of 1 to 2%w/w.

The amount of particles included within the biomaterial of the inventionmay depend on the final application of the biomaterial and of thedesired effect.

According to a preferred embodiment, the biomaterial of the invention isa chitosan or chitosan derivative gel, including 1 to 5% of chitosanparticles or 1 to 5% of chitin particles. According to anotherembodiment, the biomaterial of the invention is a chitosan derivativegel, including 1 to 2% chitin particles. In a particularly preferredembodiment, the biomaterial of the invention is a gel consistingessentially of a chitosan derivative and water with chitin and/orchitosan particles suspended in the gel, where the gel is essentiallyfree of any other formulation-enhancing agents such as plasticizers,surfactants, viscosity modifiers, and the like.

According to another embodiment of the invention, the particles includedwithin the biomaterial of the invention have a mean diameter of about 3to 150 μm, preferably 5 to 40 μm. According to an embodiment, the meandiameter of the particles are 3 to 12 μm, and preferably of 5 to 10 μm.According to another embodiment, the mean diameter of the particles are10 to 32 μm. Preferably, the particles are microspheres.

Furthermore, these bioresorbable particles in suspension in thebiomaterial of the invention should have a diameter such that thesyringability of the product using 27G (or possibly 30G) needles remainssatisfactory.

According to an embodiment, chitin and/or chitosan particles areobtained from chitin or chitosan crystals, having an averagegranulometry at the outset of 200 to 300 μm. The granulometry is reducedby any suitable technique known by one skilled in the art to lower theparticle size of the particles, such as for example, but notlimitatively, spray drying or micronization, optionally repeated morethan once. These particles may then undergo a successive series ofmicronizations, while avoiding cryomicronization which sometimes damagesthe integrity of the micronized molecules. Subsequent sifting stepseliminate those particles which have a granulometry which is either toolarge or too small.

According to an embodiment, the particles of the invention do notcontain polymethacrylic acid and/or ester derivative thereof containinghydroxyl group, polyacrylamide, polymethacrylamide,poly-N-vinyl-2-pyrrolidone, polyvinyl alcohol.

According to another embodiment, the particles are not composite, butmade of a single ingredient, which is preferably chitin.

According to an advantageous embodiment, the biomaterial of theinvention has a pH which is compatible with dermatological use,preferably a pH between 6.5 and 7.5, and ideally between 6.8 and 7.2.

According to another embodiment of the invention, the density of thebiomaterial of the invention is comparable to that of the particles,preferably between 0.95 and 1.20, and ideally between 1.00 and 1.10.

The particles may be maintained in suspension by the viscosity of theparticle-containing gel, the natural surfactant effect of chitin andchitosan, and also through the small size of the particles and the factthat their density is more or less equal to that of the gel. Thishomogeneity of density, the surfactant properties of chitin and chitosanand the small particle size ensures satisfactory homogeneity of the gel,avoiding clump formation which may block the fine needles, and avoidingthe need for additional formulation-modifying agents such asplasticizers, surfactants, and viscosity modifiers.

The invention also details a process for manufacturing a biomaterialcomprising an injectable bioresorbable polysaccharide compositioncontaining resorbable chitin and/or chitosan particles in suspension,the said process involving steps in which chitosan or chitosanderivative with a degree of deacetylation of about 30 to about 95%,preferably about 70 to about 90%, more preferably about 75 to about 85%,even more preferably about 80 to about 85%, and most preferably about85% is dissolved, followed by successive addition of glutamic acid andthen succinic anhydrid, and neutralization. The addition of particlescontaining chitin and/or chitosan under agitation may be performed atvarious stages in the process, for example before or after addition ofglutamic acid or at the end of the process.

During the neutralization step, the pH of this biomaterial is adjustedto somewhere between 6.5 and 7.5, ideally between 6.8 and 7.2, byaddition of a base such as sodium hydroxide or triethanolamine.

The resulting biomaterial may not be affected by either pH ortemperature. The latter is of particular interest as this means theproduct remains stable when stored at room temperature.

The process of manufacturing according to the invention preferably alsoincludes a sterilization step, such as a step involving irradiation orsteam sterilization for example.

According to an embodiment of the invention, the chitin and the chitosanused for manufacturing the biomaterial of the invention and/or theparticles are from one source, which is preferably GMP-grade chitosan.According to another embodiment, the manufacturing process of the chitinparticles suspended in the biomaterial uses chitin obtained byreacetylation of a GMP-grade chitosan. According to an embodiment of theinvention, the chitin used in the manufacturing process of thecomposition and/or of the particles is essentially free of protein.

The invention also relates to a medical device containing the saidbiomaterial. According to a specific embodiment of the medical device,the said biomaterial is ready-to-use in a sterile syringe.

Another aspect of this invention relates to a method for aestheticrepair treatment, in which the said biomaterial is injected by asubcutaneous or intradermal route to fill a cavity of the face or bodyin either humans or animals. More specifically, a major application ofthe invention is facial restructuring for patients suffering fromlipoatrophy, in particular those patients suffering from the HIV virus.

Lipoatrophy is characterized by a wasting of subcutaneous adiposetissue. In the face, wasting of the cheeks fat pads brings about thisvery characteristic morphological change. These fat pads are situated inthe middle region of the cheek with various extensions in the facialregion which can be affected by the fairly marked wasting of fattytissue. In particular, the main loss may be seen in the sinus extensionwhich gives a very sunken appearance to the face, although very oftenthere is also characteristic sinking of the temporal region. Thisatrophy is also very often accompanied by a wasting of retro-orbitaladipose tissue, resulting in enophthalmia which further exacerbates thefacial deterioration. Lipoatrophy results in a very degrading unwell ormorbid physical appearance in patients who, paradoxically, tend to havea satisfactory immune and virological status.

For this reason, patients oblivious to the efficacy of triple therapymay only consider the destructive effects on their physical appearanceand may not think twice about jeopardizing their treatment, failing totake the treatment properly or at the very least taking it with a greatdeal of apprehension and despair.

Another object of the invention is the use of the said biomaterial as aninjectable implant in aesthetic treatment, to fill wrinkles, fine lines,skin depressions, acne scars and other scars.

Another subject of the invention is a method of aesthetic treatment tofill a cavity in the human face or body, which may involve severalsuccessive injections of the said biomaterial, particularly in the caseof lipoatrophy, whereby each injection could be followed by massage ofthe surface of the skin over the injection area.

Another subject of the invention is the use of the said biomaterial tofill gingiva cavities or as a gingival filler.

Another subject of the invention is the use of the said biomaterial inmanufacturing a medical device aimed to be implanted in personssuffering from stress urinary incontinence alone or in association withother forms of incontinence. The said biomaterial is therefore used tofill a deficient urethral sphincter which occurs in 10 to 15% of activewomen suffering from a urinary incontinence syndrome. In the presentabsence of any satisfactory treatment, this particle gel would fulfill aspecial role as a very non-invasive treatment compared with the surgicalprocedures currently available. This method is non-aggressive,reproducible, without side-effects and only involves a local anaestheticfollowed by the para-urethral injection of the particle gel through thevaginal wall or above the urethra between the urethra and the pubic bone(an injection of around 1 to 4 cc).

The invention will be better understood upon reading the exampledetailed below, which illustrates in a non-limiting manner particularembodiments of the invention:

Example 1

Gel containing 4% of CHITOSAN (w/w)

Chitosan, GMP crustacean source, degree of deacetylation 85%, intrinsicviscosity of approx 150 cps (in a 1% acetic acid solution) was dissolvedin purified water. Glutamic acid was added in stoicchiometric quantity(according to DDA) in the solution, which, after 15 to 20 minutes,produced chitosan glutamate. Succinic anhydride was then added (samequantity as glutamic acid) yielding a gel in the form ofsuccino-chitosan glutamate. The pH of the gel was adjusted to 6.8-7.2with sodium hydroxide. Gel was then filtered through a 160 μm filter toeliminate any possible undesired particle. Purified water was then addedso as to obtain a 4% concentration of pure Chitosan in the gel. Gelobtained had a viscosity of approx 2500 cps, and was easy to injectthrough a 30 gauge needle. It was not sensitive to pH or temperature.

Example 2 Gel Containing 2% of CHITOSAN, in which 1% CHITIN Microspheres(w/w) were in Suspension

A gel was prepared in the same way as in example 1, except for theconcentration which was adjusted to 2% of pure CHITOSAN (w/w).Simultaneously, Chitosan was dissolved in a 1% acetic acid solution, andethanol was added in a proportion of 30% of the final solution. A Büchitype spray-dryer was then used in order to obtain Chitosan microspheres,with a granulometry of 5 to 13 μm. These microspheres were poured intoan acetic solution (stoicchiometric quantity of acetic acid calculatedon DDA, so as to be able to reacetylate 25 to 30% of the polymer, so asto obtain more than 50% final acetylation). The resulting chitinmicrospheres were then incorporated into the gel so as to have 1%microspheres (w/w). The final colloidal suspension had a viscosity ofapprox 3500 cps, which made it easy to inject through a 27 gauge needle,and was not sensitive to pH or temperature.

Example 3 Gel Containing 5% of CHITOSAN, in which 1% CHITIN MicronizedParticles (w/w) Wherein Suspension

A gel was prepared in the same way as in example 1, except for theconcentration which was adjusted to 5% of pure CHITOSAN (w/w). GenuineCHITIN was obtained from the GMP Chitosan supplier. The granulometry ofthis powder was 200-300 μm. The powder was micronized and sieved so asto obtain a powder with a granulometry of 5 to 32 μm. Chitin particleswere then incorporated into the gel so as to have 1% suspension in thegel. The final suspension had a viscosity of approx 4500 cps, and it wasstill possible to inject it through a 27 gauge needle. The final productwas not sensitive to pH or temperature.

1. An injectable gel comprising (a) a gel portion and (b) solidresorbable powder particles in suspension in the gel portion; (i)wherein the solid resorbable powder particles consist essentially ofchitosan powder; (ii) wherein the injectable gel comprises from 0.1 wt.% to 10 wt. % of the solid resorbable powder particles by total weightof the injectable gel; (iii) wherein the gel portion comprises from 0.1wt. % to 20 wt. % of a succinylated chitosan by total weight of theinjectable gel; (iv) wherein the injectable gel has a viscosity that issuited for subcutaneous or intradermal injection into a human.
 2. Theinjectable gel of claim 1, wherein the injectable gel has a pH of from6.5 to 7.5.
 3. The injectable gel of claim 1, wherein the injectable gelhas a pH of from 6.8 to 7.2.
 4. The injectable gel of claim 1, whereinthe succinylated chitosan is succinochitosan glutamate.
 5. Theinjectable gel according to claim 1, wherein the solid resorbable powderparticles have a mean diameter of about 3 to about 150 μm.
 6. Theinjectable gel according to claim 1, wherein the solid resorbable powderparticles have a bioresorption time of 1 to 6 months.
 7. A medicaldevice containing the injectable gel of claim
 1. 8. A medical deviceaccording to claim 7, in which the injectable gel is ready-to-use in asterile syringe.
 9. An injectable gel comprising (a) a gel portion and(b) solid resorbable powder particles in suspension in the gel portion;(i) wherein the solid resorbable powder particles consist essentially ofa mixture of chitin and chitosan powder; (ii) wherein the injectable gelcomprises from 0.1 wt. % to 10 wt. % of the solid resorbable powderparticles by total weight of the injectable gel; (iii) wherein the gelportion comprises from 0.1 wt. % to 20 wt. % of a succinylated chitosanby total weight of the injectable gel; (iv) wherein the injectable gelhas a viscosity that is suited for subcutaneous or intradermal injectioninto a human.
 10. The injectable gel of claim 9, wherein the injectablegel has a pH of from 6.5 to 7.5.
 11. The injectable gel of claim 9,wherein the injectable gel has a pH of from 6.8 to 7.2.
 12. Theinjectable gel of claim 9, wherein the succinylated chitosan issuccinochitosan glutamate.
 13. The injectable gel according to claim 9,wherein the solid resorbable powder particles have a mean diameter ofabout 3 to about 150 μm.
 14. The injectable gel according to claim 9,wherein the solid resorbable powder particles have a bioresorption timeof 1 to 6 months.
 15. A medical device containing the injectable gel ofclaim
 9. 16. A medical device according to claim 15, in which theinjectable gel is ready-to-use in a sterile syringe.